Methods and Compositions for Somatic Cell Proliferation and Viability

ABSTRACT

Methods and compositions for somatic cell proliferation as well as increasing viability of somatic cells are provided. The compositions include heparin binding protein isolated from a medium conditioned by growth of pluripotent stem cells, such as, human embryonic stem cells, human embryonic carcinoma cells. The methods include contacting a somatic cell with a heparin binding protein composition for a sufficient period of time to provide for enhanced proliferation and/or viability of the somatic cell as compared to the absence of the heparin binding protein composition.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. §119(e), this application claims priority to thefiling date of

U.S. Provisional Application No. 61/828,543, filed May 29, 2013, thedisclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.AG02725201 awarded by National Institutes of Health. The government hascertain rights in the invention.

INTRODUCTION

Unlike embryonic stem cells, somatic cells have a limited capacity forregeneration. While, embryonic stem cells have generated considerableinterest, the stem cells resident in differentiated tissues may alsoprovide an important source of regenerative capacity. These somatic, oradult, stem cells are undifferentiated cells that reside indifferentiated tissues, and have the properties of self-renewal andgeneration of differentiated cell types. The differentiated cell typesmay include all or some of the specialized cells in the tissue. Forexample, hematopoietic stem cells give rise to all hematopoieticlineages, but do not seem to give rise to stromal and other cells foundin the bone marrow.

Sources of somatic stem cells include bone marrow, blood, the cornea andthe retina of the eye, brain, skeletal muscle, dental pulp, liver, skin,the lining of the gastrointestinal tract, and pancreas. Progenitor orprecursor cells are similar to stem cells, but are usually considered tobe distinct by virtue of lacking the capacity for self-renewal.

Muscle tissue in adult vertebrates regenerates from reserve myoblastscalled satellite cells. Satellite cells are distributed throughoutmuscle tissue and are mitotically quiescent in the absence of injury ordisease. Following recovery from damage due to injury or disease or inresponse to stimuli for growth or hypertrophy, satellite cells reenterthe cell cycle, proliferate and enter existing muscle fibers or undergodifferentiation into multinucleated myotubes, which form new musclefiber. The myoblasts ultimately yield replacement muscle fibers or fuseinto existing muscle fibers, thereby increasing fiber girth.

Although, stem cells and progenitor cells present in organs of an adultorganism can proliferate and lead to generation of new somatic cells toreplace older, injured, diseased, dying, or dead somatic cells, the stemcells and progenitor cells may lose this capacity due to a number ofcauses.

With aging and in certain disease conditions, old differentiated cellslose their functionality or die, while stem and progenitor cellsdedicated to generating new differentiated cells lose their regenerativecapacity.

With explantation and in vitro culture, adult stem and progenitor cellslose stem cell function and proliferative capacity, and suffer highmortality when reintroduced in vivo.

As such, there is an interest in methods and compositions that mayincrease proliferation of somatic cells and/or increase their viability.

SUMMARY

Methods and compositions for somatic cell proliferation as well asincreasing viability of somatic cells are provided. The compositionsinclude heparin binding protein(s) isolated from a medium conditioned bygrowth of human embryonic stem cells. The methods include contacting asomatic cell with a heparin binding protein composition for a sufficientperiod of time to provide for enhanced proliferation and/or viability ofthe somatic cell as compared to the absence of the heparin bindingprotein composition.

In certain embodiments, a method for enhancing proliferation of asomatic cell is provided. The method may include contacting a somaticcell with heparin binding protein composition, wherein the compositionincludes heparin binding protein isolated from a medium conditioned bygrowth of human embryonic stem cells, where the contacting is for aperiod of time sufficient to provide for enhanced proliferation of thesomatic cell compared to the absence of the heparin binding proteincomposition.

In certain cases, the somatic cell may be a cell of an aged subject. Incertain cases, the somatic cell, e.g., a cell of an aged subject, may bean injured cell. The injury may be caused by a disease. In certaincases, the somatic cell, e.g., a cell of an aged subject, may be adiseased cell, or an explanted adult stem cell or an explantedprogenitor cell.

In certain embodiments, the somatic cell may be a muscle cell, e.g., askeletal muscle cell, a myoblast, a satellite cell, or an activatedsatellite cell. The muscle cell may be a cell of an aged subject, aninjured cell, a cell injured due to disease, a diseased cell, a cell ofan aged subject that is injured, e.g., due to a disease, or is adiseased cell, or an explanted adult stem cell or an explantedprogenitor cell.

In certain embodiments, the somatic cell may be a neural cell, e.g.,neural stem cell, neural progenitor cells, or a neuron. The neural cellmay be a cell of an aged subject, an injured cell, a cell injured due todisease, a diseased cell, a cell of an aged subject that is injured,e.g., due to a disease, or is a diseased cell, or an explanted adultstem cell or an explanted progenitor cell.

In certain embodiments, the heparin binding protein composition mayinclude a plurality of heparin binding proteins isolated from a mediumconditioned by growth of human embryonic stem cells.

Also provided herein, is use of a heparin binding protein composition,wherein the composition includes heparin binding protein isolated from amedium conditioned by growth of human embryonic stem cells, forenhancing proliferation of a somatic cell by contacting the somatic cellwith the heparin binding protein composition for a period of timesufficient to provide for enhanced proliferation of the somatic cell ascompared to the absence of the heparin binding protein composition.

The present disclosure also provides a method of increasing viability ofa neuron. The method may include contacting the neuron with heparinbinding protein composition, wherein the composition includes heparinbinding protein isolated from a medium conditioned by growth of humanembryonic stem cells, wherein the contacting is for a period of timesufficient to provide for increasing viability of the neuron as comparedto the absence of the heparin binding protein composition.

The neuron may be a cortical neuron, for example, a glutamatergicneuron.

In certain cases, the neuron may be exposed to a toxin, such as, anamyloid beta globulomer.

Also provided herein, is use of a heparin binding protein composition,wherein the composition includes heparin binding protein isolated from amedium conditioned by growth of human embryonic stem cells, forincreasing viability of a neuron by contacting the neuron with theheparin binding protein composition for a period of time sufficient toprovide for increasing viability of the neuron as compared to theabsence of the heparin binding protein composition.

Methods are also provided for screening of other agents that can improvesomatic cell proliferation and/or increase viability of somatic cells inthe presence of heparin binding protein composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Both mTeSR-1 and hESC-Conditioned mTeSR-1 increase primarymyoblast and satellite cell Proliferation and inhibit Differentiation.(A) Primary Mouse Myoblasts were cultured for 24 hours in 50%fusion/differentiation medium (DMEM, 2% horse serum) plus 50% of thespecified medium. A 2 hour BrdU pulse was performed before cell fixationto label proliferating cells. Immunofluorescence was performed for eMyHC(green) and BrdU (red), with Hoechst (blue) labeling all nuclei.Representative images are shown. Proliferation and differentiation offusion-competent myoblasts were quantified by cell scoring in 25 randomfields of each condition using a Molecular Devices automated imager andMetaXpress cell scoring software. Results are displayed as the meanpercent of BrdU+ (B) or eMyHC+ (C) proliferating or differentiatingcells +/−SD, respectively. N=4*P<4×10⁻¹⁰ for BrdU+ myoblasts incubatedin 50% mTeSR-1 as compared to myoblasts incubated in just fusion medium,or 50% hESC-conditioned mTeSR-1 as compared to myoblasts incubated injust fusion medium. *P<0.005 for eMyHC+ fusing myoblasts in 50% mTeSR-1as compared to myoblasts incubated in fusion medium alone, and *P<9×10⁻⁵for eMyHC+ fusing myoblasts in 50% hESC-conditioned mTeSR-1 as comparedto myoblasts incubated in just fusion medium. (D) Old injury activatedmyofiber-associated satellite cells were isolated at 3 days postcardiotoxin-induced muscle injury, and cultured overnight in 50%DMEM/F12 with 10% old serum, and 50% of the medium specified, followedby a 2 hour BrdU pulse to label proliferating cells before cellfixation. Immunofluorescence was performed with Desmin (green) and BrdU(red), with Hoechst (blue) labeling all cell nuclei. Representativeimages are shown and demonstrate that both hESC-conditioned mTeSR-1 andmTeSR-1 have a pro-myogenic effect on activated satellite cells. (E)Proliferating Desmin+/BrdU+ satellite cells were quantified by cellscoring in multiple random fields of each condition. Results aredisplayed as the mean percent of BrdU+/Desmin+ proliferating satellitecell cells +/−SD. N=3, *P<0.05 for satellite cells in 50% mTeSR-1 ascompared to satellite cells incubated in just basal medium with oldserum, and *P<0.001 for satellite cells in 50% hESC-conditioned mTeSR-1as compared to satellite cells incubated in just basal medium with oldserum. (F) Undifferentiated hESCs that were grown in mTeSR-1 medium werewashed 0-3 times with Opti-MEM, followed by overnight incubation inOpti-MEM and collection of the resulting conditioned Opti-MEM. ThehESC-conditioned Opti-MEM was spun down to remove cell debris, beforeaddition to myoblasts as a 50/50 mix with myogenic fusion medium forculture overnight. A 2 hour BrdU pulse was performed to labelproliferating cells prior to cell fixation and immunofluorescence wasperformed with eMyHC and BrdU, with Hoechst labeling all cell nuclei(images not shown). Proliferating and differentiating cells werequantified by cell scoring 25 random fields of each condition using anautomated imager and MetaXpress cell scoring software. Results aredisplayed as the mean percent of BrdU+ or eMyHC+ proliferating ordifferentiating cells +/−SD, respectively. N=2

FIG. 2. hESC-conditioned medium enhances myogenic proliferation in theabsence of FGF2 in mTeSR-1 growth medium. (A) Primary myoblasts werecultured for 16 hours in 50% fusion/differentiation Medium+50% of thespecified medium. A 2 hour BrdU pulse was performed before cell fixationto label proliferating cells. Immunofluorescence was performed for eMyHC(green) and BrdU (red), with Hoechst (blue) labeling all nuclei.Representative images demonstrate that hESC-conditioned medium lackingFGF2 increases myoblast proliferation and inhibits differentiation. (B)Proliferation and differentiation of fusion-competent myoblasts werequantified by cell scoring in 50 random fields of each condition usingan automated imager and MetaXpress cell scoring software. Results aredisplayed as the mean percent of BrdU+ or eMyHC+ proliferating ordifferentiating cells +/−SD, respectively. N=4, *P<2×10⁻¹² forhESC-conditioned basal medium with 4 mTeSR-1 ingredient components(lacking FGF2) as compared to differentiation hESC-conditioned basalmedium with 4 mTeSR-1 ingredient components (also lacking FGF2), and forhESC-conditioned basal medium with 4 mTeSR-1 ingredient components(lacking FGF2) as compared to myoblasts incubated in basal medium with 4mTeSR-1 ingredient components (lacking FGF2).

FIG. 3. Age-dependent comparison of FGF2 and pERK levels andlocalization in muscle fibers. (A) Protein was isolated fromfreshly-derived uninjured myofibers of young and old mice and the levelsof FGF2 and phospho-ERK1/2; total ERK1/2 and cytoplasmic beta-actin wereanalyzed by Western blotting, using specific antibodies. Representativedata are shown. (B) Relative protein expression was quantified in 3young and 3 old mice by normalization of FGF-2 to beta-actin andnormalization of pERK to total ERK; significantly higher levels ofFGF-2, but not of pERK were detected in the old myofibers, as comparedto young (n=3, * P<0.05). (C) Tibialis anterior (TA) muscle from 2 youngand 2 old mice were sectioned and immunostained for laminin (green) andFGF2 (red). Hoechst (blue) labels all nuclei. Representative imagesdemonstrate the presence of FGF-2 and laminin in muscle compartments, ascompared to the negative IgG control and higher FGF-2 levels seem to bepresent in the laminin+ basement membranes of the young myofibers, ascompared to old. (D). The pixel density of FGF-2 that co-localizes withlaminin+ basement membrane vs. the internal regions of the myofibers wasdetermined in 30-40 areas of each cryosection of 3 muscle tissue slidesfrom young and old muscle, using Image J software. Preferentiallocalization of FGF-2 in the basement membrane was identified in youngmuscle, while in the old tissue, FGF-2 was mis-localized to the centerof the myofibers and away from the basement membrane, n=3, *P<0.05.

FIG. 4. Age-related comparison of FGF2 and pERK levels in muscle stemcells derived from uninjured tissue and of proliferation of these cells.(A) Quiescent muscle stem cells were isolated from uninjured young andold muscle as described in Methods. The cells were treated (or not) withFGF2 (10 ng/ml) for 30 minutes before being lysed and analyzed for thelevels of FGF2, phospho-ERK1/2, total ERK1/2 and beta actin by WesternBlotting. Representative images are shown. (B) Relative proteinexpression of FGF-2, pERK and total ERK were quantified from 3 young and3 old mice, using beta-actin for normalization. The levels of FGF-2 wereequally undetectable in young and old satellite cells, however, addedFGF-2 was clearly detected in the cells of both ages after ˜2minexposure (but was not detected in accelular samples even after 10 minexposure); the levels of pERK and total were equally low in young andold satellite cells and pERK, but not total ERK was, as expected,induced by added FGF-2. n=3, *P<0.05. (C-E) Muscle stem cells fromresting muscle were treated (or not) with FGF2 (10 ng/ml) for 24 hoursbefore immunostaining for Ki67 and Pax7. Percent of Ki67+/Pax7+proliferating myogenic cells were quantified. No age-specific increasein cell proliferation was detected in satellite cells isolated from olduninjured muscle, and in contrast, more proliferating satellite cellswere observed in the cultures derived from young muscle. Added FGF-2enhanced the proliferation of both young and old muscle stem cells inthese overnight cultures. n=3, *P<0.05.

FIG. 5. Expression of FGF2 in quiescent muscle stem cells from young andold mice. Muscle stem cells were isolated from young and old uninjuredmuscle, as described in Methods. The cells were immediately lysed forWestern blotting without culturing and the expression of FGF2,phosphor-ERK1/2, ERK1/2 and β-actin were analyzed. 30 minutes ofenhanced chemiluminescence exposure was used for detection of FGF-2,while pERK, total ERK and actin were detected after 2 min, 30 sec and 30sec exposure, respectively. Low and age-independent levels of FGF-2 andpERK were detected in satellite cells that were derived from uninjuredyoung and old TA muscle.

FIG. 6. Myogenic marker expression in young and old muscle stem cells.

Muscle stem cell isolated from young and old mice were cultured for 24hours and then immunostained for myogenic markers Pax7 and Myf-5. ˜95%of isolated young and old satellite cells expressed these myogenicmarkers, demonstrating high and age-independent purity.

FIG. 7. Proteinase K treatment abolishes proliferative hESC factors. Oldinjury-activated satellite cells with associated myofibers were culturedovernight in 50% Opti-MEM with 10% old serum and 50% hESC conditionedOpti-MEM that was treated with pre-washed Proteinase K agarose beads(Sigma-Aldrich), for 1 hour at 37 C followed by bead removal, ormock-treated hESC conditioned Opti-MEM. Cells received a 2 hour BrdUpulse to label proliferating cells before cell fixation.Immunofluorescence was performed for Desmin (green) and BrdU (red), withHoechst (blue) labeling all cell nuclei. Proliferating, desmin+ve cellswere quantified by imaging and scoring multiple random microscopicfields of each condition. Results are displayed as the mean percent ofBrdU+,Desmin+ proliferating satellite cell cells +/−SEM, p<0.005, n=3replicate experiments.

FIG. 8. Pro-regenerative Embryonic Factors Contain Heparin BindingDomains. (A) Primary Mouse Myoblasts were cultured for 24 hours in 50%fusion/differentiation medium+50% of the specified medium. A 2 hour BrdUpulse was performed before cell fixation to label proliferating cells.Immunofluorescence was performed for eMyHC (green) and BrdU (red), withHoechst (blue) labeling all nuclei. Representative images are shown. (B)Proliferation and differentiation of fusion-competent myoblasts werequantified by cell scoring in 25-100 random fields of each conditionusing an automated imager and MetaXpress cell scoring software. Resultsare displayed as the mean percent of BrdU+ or eMyHC+ proliferating ordifferentiating cells +/−SD, respectively. N=6*P<3×10^(̂−45) forhESC-conditioned Opti-MEM compared to differentiated hESC-conditionedOpti-MEM, and hESC-conditioned Opti-MEM compared to Opti-MEM. *P<0.005for hESC-conditioned Opti-MEM compared to heparin depletedhESC-conditioned Opti-MEM, and hESC-conditioned Opti-MEM compared todifferentiated hESC-conditioned OptiMEM. *P<5×10^(̂−7) forhESC-conditioned Opti-MEM compared to Opti-MEM, and Eluted Proteinscompared to Opti-MEM. (C) Old Tibialis Anterior muscles were injuredwith cardiotoxin (see Methods). Heparin bound and eluted protein orvehicle control (Opti-MEM) were injected into sites of injury on Day 0and Day 2. BrdU was injected (intraperitoneal) at 3 days post injury tolabel proliferating, fusion-competent myoblasts. Animals were sacrificedand muscle was collected 5 days post injury. Cryosections (10 μm) wereanalyzed by hematoxylin/eosin (H&E) staining and immunostaining forembryonic myosin heavy chain (eMyHC, shown in green) and BrdUincorporation (shown in red). Hoechst stains nuclei (blue). As shown byrepresentative images, the regenerative outcome of old muscle giveneluted factors was significantly improved as compared to old musclegiven Opti-MEM vehicle control, based on significantly diminished scartissue formation, larger and more dense de novo myofibers and anincrease in the numbers of eMyHC+ myofibers with centrally-located BrdU+nuclei that replaced the damaged tissue. (D) Regeneration of old mouseTibialis Anterior 5 days post injury, that received eluted factors orvehicle, was quantified from muscle sections, and is presented as thenumber of newly regenerated myofibers per square millimeter of injurysite. Error bars indicate SD, n=3 mice per group. *P<0.02 between oldgiven eluted factors and old given vehicle control.

FIG. 9. hESC-secreted Factors Enhance NPC Proliferation and areNeuroprotective. (A) Rat Neural Progenitor Cells (rNPCs) were culturedovernight in 50% differentiation medium (DMF12+N2) and 50% specifiedmedium followed by a 4 hour BrdU pulse to label proliferating cellsprior to fixation. Immunofluorescence was performed for Sox2 (red) andBrdU (green), with Hoechst (blue) labeling cell nuclei. Representativeimages are shown. (B) Quantification of BrdU+/Sox2+ proliferating cellswas performed by cell scoring in 100 random fields of each conditionusing an automated imager and MetaXpress cell scoring software. Resultsare displayed as the mean percent of BrdU+/Sox2+ proliferating cells+/−SD; N=4, *P<2×10⁻¹⁵ for rNPCs incubated in hESC-conditioned Opti-MEMas compared to rNPCs incubated in differentiated hESC-conditionedOpti-MEM, and *P<0.0002 for rNPCs incubated in hESC-conditioned Opti-MEMas compared to rNPCs incubated in Opti-MEM. (C) Pre-incubation of Aβglobulomers with hESC-conditioned Opti-MEM before incubation with maturecortical neurons prevents neuron cell death and exhibits a neurotrophiceffect, as shown via decreased immunofluorescence staining of cleavedcaspase3 (red) and increased Map2+ (green) neurons. Hoechst (blue)labels all nuclei. Representative images are shown. (D) Total number ofMap2+ neurons and the amount of apoptosis was quantified by cell scoringof random fields taken by an automated imager of each condition in theabove assay performed in replicates. Results are displayed as the meanpercent of caspase+ or Map2+ (C) proliferating or differentiating cells+/−SD, respectively. N=4, *P<0.02 for Map2+ cortical neurons treatedwith Aβ globulomers preincubated in hESC-conditioned Opti-MEM, ascompared to treatment with Aβ globulomers in OptiMEM, and *P<0.05 forthe level of caspase3 in cortical neurons treated with Aβ globulomerspreincubated in hESC-conditioned Opti-MEM, as compared to treatment withAβ globulomers in Opti-MEM.

DEFINITIONS

By “embryonic stem cell” or “ES cell” it is meant a cell that a) canself-renew, b) can differentiate to produce all types of cells in anorganism, and c) is derived from a developing organism or is anestablished ES cell line which was derived from a developing organism.ES cell may be derived from the inner cell mass of the blastula of adeveloping organism. ES cell may be derived from a blastomere generatedby single blastomere biopsy (SBB) involving removal of a singleblastomere from the eight cell stage of a developing organism. Ingeneral, SBB provides a non-destructive alternative to inner cell massisolation. SBB and generation of hES cells from the biopsied blastomereis described in Cell Stem Cell, 2008 Feb. 7; 2(2):113-7. ES cells can becultured over a long period of time while maintaining the ability todifferentiate into all types of cells in an organism. In culture, EScells typically grow as flat colonies with large nucleo-cytoplasmicratios, defined borders and prominent nuclei. In addition, ES cellsexpress SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase,but not SSEA-1. Examples of methods of generating and characterizing EScells may be found in, for example, U.S. Pat. No. 7,029,913, U.S. Pat.No. 5,843,780, and U.S. Pat. No. 6,200,806, the disclosures of which areincorporated herein by reference.

By “somatic cell” it is meant any cell of an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells arecells that have differentiated sufficiently that they will not naturallygenerate cells of all three germ layers of the body, i.e., ectoderm,mesoderm and endoderm. For example, somatic cells would include bothneurons and neural progenitors, the latter of which may be able toself-renew and naturally give rise to all or some cell types of thecentral nervous system but cannot give rise to cells of the mesoderm orendoderm lineages.

By “pluripotent stem cell” or “pluripotent cell” it is meant a cell thathas the ability under appropriate conditions of producing progeny ofseveral different cell types that are derivatives of all of the threegerminal layers (endoderm, mesoderm, and ectoderm) Pluripotent stemcells are capable of forming teratomas. Examples of pluripotent stemcells are embryonic stem (ES) cells, embryonic germ stem (EG) cells,embryonic Carcinoma (EC) Cells, induced pluripotent stem (iPS) cells,and adult stem cells. PS cells may be from any organism of interest,including, primate, e.g., human; canine; feline; murine; equine;porcine; avian; camel; bovine; ovine, and so on.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual”, “subject”, “host”, and “patient” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, e.g., humans.

By “endoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to the gastrointestinal tract, respiratorytract, endocrine glands and organs, certain structures of the auditorysystem, and certain structures of the urinary system.

By “mesoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to muscles, cartilage, bones, dermis, thereproductive system, adipose tissue, connective tissues of the gut,peritoneum, certain structures of the urinary system, mesothelium,notochord, and spleen.

By “ectoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to the nervous system, tooth enamel,epidermis, hair, nails, and linings of mucosal tissues.

The term “medium” in context of cell culture or the phrase “cell culturemedium” or “cell medium” or “growth medium” refer to a cellular growthmedium suitable for culturing of human ES cells. Examples of cellculture medium include Minimum Essential Medium (MEM), Eagle's Medium,Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified EagleMedium: Nutrient Mixture F-12 (DMEM/F12), F10 Nutrient Mixture, Ham'sF10 Nutrient Mix, Ham's F12 Nutrient Mixture, Medium 199, RPMI, RPMI1640, reduced serum medium, basal medium (BME), DMEM/F12 (1:1), and thelike, and combinations thereof. The medium or cell culture medium may bemodified by adding one or more additives. Additives may include serum,such as, fetal bovine serum and/or serum replacement agents, such as,B27, N2, KSR, and combinations thereof.

The term “enriched” used herein in the context of heparin bindingprotein(s) means that the fraction of heparin binding protein(s) isincreased by at least 10% over the fraction of other proteins in theculture medium.

A “pharmaceutically acceptable carrier” means carrier that is useful inpreparing a pharmaceutical composition that is generally safe, non-toxicand neither biologically nor otherwise undesirable, and include carriersthat are acceptable for veterinary use as well as human pharmaceuticaluse. A pharmaceutically acceptable carrier as used in the specificationincludes both one and more than one such carrier.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. A “pharmaceutical composition” may be sterile andfree of contaminants that are capable of eliciting an undesirableresponse within the subject (e.g., the compound(s) in the pharmaceuticalcomposition is pharmaceutical grade). Pharmaceutical compositions can bedesigned for administration to subjects or patients in need thereof viaa number of different routes of administration.

As used herein, “marker” refers to any molecule that can be measured ordetected. For example, a marker can include, without limitations, anucleic acid, such as, a transcript of a gene, a polypeptide product ofa gene, a glycoprotein, a carbohydrate, a glycolipid, a lipid, alipoprotein, a carbohydrate, or a small molecule (for example, amolecule having a molecular weight of less than 10,000 amu).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the protein”includes reference to one or more proteins and equivalents thereof knownto those skilled in the art, and so forth. It is further noted that theclaims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Methods and compositions for somatic cell proliferation as well asincreasing viability of somatic cells are provided. The compositionsinclude heparin binding protein isolated from a medium conditioned bygrowth of human embryonic stem cells, or embryonic carcinoma cells. Themethods include contacting a somatic cell with a heparin binding proteincomposition for a sufficient period of time to provide for enhancedproliferation and/or viability of the somatic cell as compared to theabsence of the heparin binding protein composition.

Methods are also provided for screening of other agents that can improvesomatic cell proliferation and/or increase viability of somatic cells inthe presence of heparin binding protein composition.

The methods and compositions are described in detail below.

Methods for Enhancing Somatic Cell Proliferation and/or Viability

As noted above, in certain embodiments, a method for enhancingproliferation of a somatic cell is provided. The method may includecontacting a somatic cell with heparin binding protein composition,wherein the composition includes heparin binding protein isolated from amedium conditioned by growth of human embryonic stem cells, or embryoniccarcinoma cells, where the contacting is for a period of time sufficientto provide for enhanced proliferation of the somatic cell compared tothe absence of the heparin binding protein composition.

The present disclosure also provides a method of increasing viability ofa somatic cell. The method may include contacting the somatic cell withheparin binding protein composition, wherein the composition includesheparin binding protein isolated from a medium conditioned by growth ofhuman embryonic stem cells, or embryonic carcinoma cells, wherein thecontacting is for a period of time sufficient to provide for increasingviability of the somatic cell as compared to the absence of the heparinbinding protein composition.

Any somatic cell may be used in the present methods, including but notlimited to, stem cell, progenitor cell, muscle cell, myoblast, satellitecell, neuron, blood cell, and the like.

The term stem cell is used herein to refer to a mammalian cell that hasthe ability both to self-renew, and to generate differentiated progeny(see Morrison et al. (1997) Cell 88:287-298). Generally, stem cells alsohave one or more of the following properties: an ability to undergoasynchronous, or symmetric replication, that is where the two daughtercells after division can have different phenotypes; extensiveself-renewal capacity; capacity for existence in a mitotically quiescentform; and clonal regeneration of all the tissue in which they exist, forexample the ability of hematopoietic stem cells to reconstitute allhematopoietic lineages. “Progenitor cells” differ from stem cells inthat they typically do not have the extensive self-renewal capacity, andoften can only regenerate a subset of the lineages in the tissue fromwhich they derive, for example only lymphoid, or erythroid lineages in ahematopoietic setting. In certain cases, the somatic cell may be anexplanted stem cell or progenitor cell. In certain cases, the explantedstem cell or progenitor cell may have been subjected to culturing for aprolonged duration of time, such as, 1 day to 10 years or more, such as,1 day, 3 days, 7 days, 2 weeks, 5 weeks, 10 weeks, 3 months, 10 months,1 year, 3 years, 5 years, 10 years, or more.

Stem cells may be characterized by both the presence of certain markersand the absence of certain markers. These markers may be detected usinga number of methods that may depend on the nature of the marker. In someembodiments, the marker may be associated with specific epitopes whichare identified by antibodies. Stem cells may also be identified byfunctional assays both in vitro and in vivo, particularly assaysrelating to the ability of stem cells to give rise to multipledifferentiated progeny.

Stem cells of interest include muscle satellite cells; hematopoieticstem cells and progenitor cells derived therefrom (U.S. Pat. No.5,061,620); neural stem cells (see Morrison et al. (1999) Cell96:737-749); mesenchymal stem cells; mesodermal stem cells; liver stemcells, etc.

The somatic cells of interest are typically mammalian, where the termrefers to any animal classified as a mammal, including humans, domesticand farm animals, and zoo, laboratory, sports, or pet animals, such asdogs, horses, cats, cows, mice, rats, rabbits, etc. Preferably, themammal is human.

In certain embodiments, the somatic cell is a muscle cell. The termmuscle cell as used herein refers to any cell that contributes to muscletissue. Myoblasts, satellite cells, myotubes, and myofibrils are allincluded in the term “muscle cells”. Muscle cell includes skeletal,cardiac and smooth muscle cells.

Activation of satellite cells in muscle tissue can result in theproduction of new muscle cells in a subject. Muscle regeneration as usedherein refers to the process by which new muscle fibers form from musclestem cells, such as, satellite cells and myoblasts.

In certain embodiments, a method for regeneration of skeletal musclecell is provided. The method may include contacting a skeletal musclecell with heparin binding protein composition, wherein the compositionincludes heparin binding protein isolated from a medium conditioned bygrowth of human embryonic stem cells, where the contacting is for aperiod of time sufficient to provide for regeneration of the skeletalmuscle cell compared to the absence of the heparin binding proteincomposition.

In certain embodiments, the skeletal muscle cell may be in a muscletissue. In certain embodiments, the muscle tissue may include satellitecells. In certain cases, the muscle tissue may be injured resulting inactivation of the satellite cells.

In certain cases, the method may include contacting an injured musclestissue with heparin binding protein composition, wherein the injuredmuscles tissue includes activated satellite cells, wherein thecomposition includes heparin binding protein isolated from a mediumconditioned by growth of human embryonic stem cells, where thecontacting is for a period of time sufficient to provide forproliferation of the activated satellite cells and result in enhancedregeneration of muscle cells in the injured skeletal muscle tissue ascompared to the absence of the heparin binding protein composition.

In certain cases, the injured muscle tissue may be of an aged subject.As used herein, the term aged refers to the effects or thecharacteristics of increasing age, particularly with respect to thediminished ability of somatic tissues to regenerate in response todamage, disease, and normal use. One measure of aging is evidenced bythe inability of the organism to provide suitable signals for theactivation of somatic stem cells. Examples of such signals are providedin U.S. Pat. No. 7,837,993. Such signals include soluble factors thatmay be empirically measured, e.g., by functional assay such as theability of soluble factors in the patient blood to induce stem cellactivation in response to tissue damage; or by the ability to induceexpression of Notch ligands, by binding assays such as ELISA, RIA, withbinding agents specific for Notch ligands; or by the ability to increasethe levels of activated Notch, with Western analysis for activated,truncated Notch.

Aging may be also be defined in terms of general physiologicalcharacteristics.

The rate of aging is very species specific, where a human may be aged atabout 50 years; and a rodent at about 2 years. In general terms, anatural progressive decline in body systems starts in early adulthood,but it becomes most evident several decades later. One arbitrary way todefine old age more precisely in humans is to say that it begins atconventional retirement age, around about 60, around about 65 years ofage. Another definition sets parameters for aging coincident with theloss of reproductive ability, which is around about age 45, more usuallyaround about 50 in humans, but will, however, vary with the individual.

In certain embodiments, the present methods may result in an increase inthe number of new skeletal muscle fibers by at least 1%, by at least 5%,by at least 10%, by at least 20%, by at least 30%, by at least 40%, orby at least 50%. The growth of muscle may occur by the increase in thefiber size and/or by increasing the number of fibers. The growth ofmuscle may be measured by an increase in wet weight, an increase inprotein content, an increase in the number of muscle fibers, an increasein muscle fiber diameter, etc. An increase in growth of a muscle fibercan be defined as an increase in the diameter where the diameter isdefined as the minor axis of ellipsis of the cross section.

Muscle regeneration may also be monitored by the mitotic index ofmuscle. For example, cells may be exposed to a labeling agent for a timeequivalent to two doubling times. The mitotic index is the fraction ofcells in the culture which have labeled nuclei when grown in thepresence of a tracer which only incorporates during S phase (e.g., BrdU)and the doubling time is defined as the average S time required for thenumber of cells in the culture to increase by a factor of two.Productive muscle regeneration may be also monitored by an increase inmuscle strength and agility and other measurements of muscle function.

In addition to skeletal muscle regeneration, the regeneration of cardiacmuscle in an aged subject is of interest. For example, following anevent such as myocardial infarction; surgery, catheter insertion,atherosclerosis, and the like, cardiac muscle can be damaged. Suchdamage is not easily repaired in elderly patients, resulting in a lossof function. Administration of a heparin binding protein composition, asdisclosed herein, following such incidents of muscle damage can increaseregeneration of the damaged tissues. The heparin binding proteincomposition may be administered systemically, or using a stent,catheter, implant, and the like that increase the local concentration ofthe heparin binding protein composition.

In certain embodiments, the contacting may include administering theheparin-binding protein composition to a subject having or at risk ofdeveloping an aging related condition. A number of conditions relevantto aged populations are characterized by an inability to regeneratetissues. All aged organs and tissues undergo a loss of regeneration andmaintenance with age, thus this method is applicable to the aged organsystems in general, including muscle, brain, blood, bones, liver, skin,etc.

Hematopoietic stem cells (HSCs) have the ability to renew themselves andto give rise to all lineages of the blood. Conditions of the aged thatbenefit from enhance proliferation of HSC include, for example,conditions of blood loss, such as surgery, injury, and the like, wherethere is a need to increase the number of circulating hematopoieticcells. Anemia is an abnormal reduction in red blood cells, which canoccur from a malfunction in the production of red blood cells. Weaknessand fatigue are the most common symptoms of even mild anemia. Anemia inthe elderly is often due to causes other than diet, particularlygastrointestinal bleeding or blood loss during surgery. Anemia in olderpeople is also often due to chronic diseases and folic acid and othervitamin deficiencies.

In conditions of the aged where there is a requirement for hematopoieticcell generation, a heparin binding protein composition may beadministered, e.g., following incidents of blood loss, and the like.

Neural stem cells are primarily found in the hippocampus, and may giverise to neurons involved in cognitive function, memory, motor control,and the like. Neural stem and progenitor cells can participate inaspects of normal development, including migration alongwell-established migratory pathways to disseminated CNS regions,differentiation into multiple developmentally- andregionally-appropriate cell types in response to micro-environmentalcues, and non-disruptive, non-tumorigenic interspersion with hostprogenitors and their progeny.

Aged individuals often suffer from a diminution of neural function. Assuch, the methods provided herein find use in the treatment of a varietyof conditions, including traumatic injury to the spinal cord, brain, andperipheral nervous system; treatment of degenerative disorders includingAlzheimer's disease, Huntington's disease, Parkinson's disease;Creutzfeldt-Jakob disease; stroke; and the like.

The methods disclosed herein may also be carried out on stem cellspresent in the epidermis, which give rise both to epidermal andmesenchymal tissues. Like most of the body's tissues, the skin undergoesmany changes in the course of the normal aging process. The cells dividemore slowly, and the inner layer of the dermis starts to thin. Fat cellsbeneath the dermis begin to atrophy. In addition, the ability of theskin to repair itself diminishes with age, so wounds are slower to heal.The thinning skin becomes vulnerable to injuries and damage. Theunderlying network of elastin and collagen fibers, which providesscaffolding for the surface skin layers, loosens and unravels. Skin thenloses its elasticity. When pressed, it no longer springs back to itsinitial position but instead sags and forms furrows. The skin is morefragile and may bruise or tear easily and take longer to heal.

In response to damage of aged skin, for cosmetic purposes, followingtrauma such as burns, abrasions, etc., it may be beneficial to stimulateproliferation of stem cells. Heparin binding protein composition may beadministered topically, e.g., in combination with agents to enhancepenetration through the dermal layers, systemically, using implants,etc.

In many clinical situations, the bone healing condition are less idealdue to decreased activity of bone forming cells, e.g., within agedpeople. Within bone marrow stroma there exists a subset ofnon-hematopoiethic cells capable of giving rise to multiple celllineages. These cells termed as mesenchymal stem cells (MSC) havepotential to differentiate to lineages of mesenchymal tissues includingbone, cartilage, fat, tendon, muscle, and marrow stroma.

A variety of bone and cartilage disorders affect aged individuals. Suchtissues are normally regenerated by mesenchymal stem cells. Included insuch conditions is osteoarthritis. Osteoarthritis occurs in the jointsof the body as an expression of “wear-and-tear”. Thus athletes oroverweight individuals develop osteoarthritis in large joints (knees,shoulders, hips) due to loss or damage of cartilage. This hard, smoothcushion that covers the bony joint surfaces is composed primarily ofcollagen, the structural protein in the body, which forms a mesh to givesupport and flexibility to the joint. When cartilage is damaged andlost, the bone surfaces undergo abnormal changes. There is someinflammation, but not as much as is seen with other types of arthritis.Nevertheless, osteoarthritis is responsible for considerable pain anddisability in older persons.

In conditions of the aged where repair of mesenchymal tissues isdecreased, or there is a large injury to mesenchymal tissues, the stemcell proliferation may be enhanced by administration of the heparinbinding protein composition.

In certain cases, the contacting may increase somatic cell proliferationby about 10% or more compared to the absence of the heparin bindingprotein composition. For example, the contacting may increase thesomatic cell proliferation by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or more compared to the absence of the heparin bindingprotein composition.

In some instances, the contacting may be administering theheparin-binding protein composition to the subject after the occurrenceof a tissue injury, e.g., 1 or more days after the injury occurrence,such as 2 or more days after the injury occurrence. The administering ofthe heparin-binding protein composition may be performed 0 days to 30days after occurrence of the injury. In certain cases, the administeringmay be performed 1 days to 25 days, or 3 days to 20 days, or 3 days to15 days, or 3 days to 10 days, or 3 days to 7 days, or after 3 days to 5days, after the occurrence of the injury.

Following delivery of heparin-binding protein composition, the subjectmay be given standard rehabilitative therapy and the repair of damagedand/or altered tissue may be assessed. Assessment of reparative orrestorative effects may include testing for physical (e.g., musclestrength, motor skills, movement of extremities, speech, etc.) and/orcognitive function by any convenient protocol over the course of one,two, four and six weeks after administration of heparin binding proteincomposition.

In certain cases, the somatic cell may be a cell that has a decreasedviability due to a number of conditions, such as, aging, injury,disease, and/or exposure to a toxin, such as, mutant huntingtin,hyperphosphorylated tau, prions; proteins aggregates, such as, anamyloid beta globulomer, polyglutamine protein aggregates, and the like.

In certain cases, the subject methods may be used to treat a subjecthaving a condition characterized by neural cell death, such as,traumatic brain injury, Alzheimer's Parkinson's, Huntington's,Creutzfeldt-Jakob, and other diseases.

In certain cases, the subject methods may be used to treat a subject atrisk of developing a condition characterized by neural cell death. Incertain cases, a subject at risk of developing a condition characterizedby neural cell death may be identified by assaying a marker(s) for thedisease, such as, presence of a mutant gene, presence of other geneticmarkers, and the like.

The somatic cell with decreased viability may be any somatic cell asprovided herein. In particular embodiments, the somatic cell may be aneuron, such as, a cortical neuron, e.g., a glutamatergic neuron.

In certain cases, a method for enhancing viability of a cortical neuronexposed to a toxin, such as, amyloid beta globulomer is provided, themethod may include contacting the cortical neuron with a heparin bindingprotein composition.

The timing of administration of the heparin binding protein compositionmay be determined on a case-by-case basis and can be for therapeuticand/or prophylactic purposes.

The heparin-binding protein composition and the routes of administrationmay be as described in the present disclosure.

In certain cases, the contacting may increase somatic cell viability byabout 2 months to about 50 years or more compared to the absence of theheparin binding protein composition. For example, the contacting mayincrease the somatic cell viability by 2 months, 6 months, 1 year, 3years, 5 years, 8 years, 10 years, 15 years, 20 years, 25 years, 30years, 35 years, 40 years, 45 years, 50 years, 55 years or more comparedto the absence of the heparin binding protein composition.

The increased viability of a somatic cell may refer to prevention ofloss of the cell as evidence by necrosis or apoptosis. Increase insomatic cell viability may be assayed by any method known in the art andmay include assessing function of the somatic cell. For example, themethod may include administering the heparin binding protein compositionto a subject having or at risk of developing a neural disorder that hasa specific set of symptoms. Assessing of increased neural cell viabilitywould include monitoring the absence of development of the symptoms,where complete absence or delayed development of symptoms would indicatethat the administration of the heparin binding protein composition iseffective in increasing cell viability. In cases where the symptoms haveappeared, the assessing would include monitoring the symptoms, where adecrease in the severity of the symptoms, a lack of or decreased rate ofincrease in the severity of the symptoms, or disappearance of thesymptoms indicate that the administration of the heparin binding proteincomposition is effective in increasing cell viability.

In a specific embodiment, the heparin-binding protein composition can beused for treatment of patients by means of a short-term administration,e.g., of 1, 2, 3 or more days, up to 1 or 2 weeks, in order to obtain arapid, significant increase in somatic cell proliferation. In anotherembodiment, the heparin-binding protein composition can be used fortreatment of patients by means of a long-term administration, e.g., oncea month, every two months, every three months, every three months, everysix months every nine months, every year, every three years, or lessfrequently for the life time of the patient.

As effective amount of the heparin binding protein composition in themethods provided here may be determined empirically, for example, usinganimal models as provided herein. In vitro models are also useful forthe assessment of effective amount. For example, cultures are describedherein where the regenerative potential of stem cells are evaluated inthe absence or presence of heparin binding protein composition.

Contacting of the somatic cell with the heparin binding proteincomposition may be carried out by a variety of appropriate methods. Incertain embodiments, the heparin binding protein composition may beadministered to a subject. The administering may be via any appropriateroute, including systemic or localized routes. Routes of administrationmay be combined, if desired, or adjusted depending upon thepharmaceutical composition and/or the desired effect.

Also disclosed herein is a culture medium enriched for heparin bindingproteins, where the culture medium is medium conditioned by growth ofhuman embryonic stem cells. Also disclosed herein are methods forenhancing proliferation or viability of a somatic cell, where the methodincludes contacting the somatic cell with a culture medium enriched forheparin binding proteins, where the culture medium is medium conditionedby growth of human embryonic stem cells.

Also provided herein are methods for enhancing viability and/orproliferation of adult stem cells and/or adult progenitor cells invitro. With explantation and in vitro culture, adult stem and progenitorcells lose stem cell function and proliferative capacity, and sufferhigh mortality when reintroduced in vivo. As such, the subject methodsmay be used to enhance proliferation and viability of explanted adultstem and progenitor cells by contacting the explanted adult stem oradult progenitor cells with heparin binding protein composition, whereinthe composition includes heparin binding protein isolated from a mediumconditioned by growth of human embryonic stem cells, or embryoniccarcinoma cells, where the contacting is for a period of time sufficientto provide for enhanced proliferation of the explanted adult stem oradult progenitor cells compared to the absence of the heparin bindingprotein composition.

Routes of Administration

The heparin-protein binding composition or a cell culture mediumenriched for heparin binding proteins secreted by human ES or embryoniccarcinoma cells may be administered using any medically appropriateprocedure, e.g., intravascular (intravenous, intraarterial,intracapillary) administration, injection into tissue, injection intocerebrospinal fluid, intracavity injection, or the like.

Where local delivery is desired, administration may involveadministering the composition to a desired target tissue, such asmuscle, brain, spine, etc. For local delivery, the administration may beby injection or by placement of the composition in the desired tissue ororgan by surgery, for example. In certain cases, an implant, such as acannula implant, that acts to retain the active dose at the site ofimplantation may be used.

In some instances, hydrogel delivery may be employed, e.g., as describedin Piantino J, et al. (2006) Exp Neurol. 201:359-67; and Ma J, et al.(2007) Biomed Mater. 2:233-40. In some instances, systemic,intraperitoneal, intravascular or subcutaneous protocols are employed,e.g., as described in Pardridge W M (2008) Bioconjug Chem. 19:1327-38.In some instances, nanoparticle mediated delivery protocols may beemployed, e.g., as described in Tosi G, et al. (2008). Expert Opin DrugDeliv. 5:155-74; and Ulbrich K, et al. (2008); Eur J Pharm Biopharm.2008 Sep. 5. In some instances, intracerebral, ventricular orintrathecal delivery protocols may be employed, e.g., as described inBuchli A D and Schwab M E (2005) Ann Med. 37:556-67; and Shoichet M S,et al. (2007) Prog Brain Res. 161:385-92. In some instances, intranasaldelivery protocols are employed, e.g., as described in Smith PF (2003)IDrugs. 6:1173-7; and Vyas TK, et al. (2006) Crit Rev Ther Drug CarrierSyst. 23:319-47.

Intrathecal administration may be carried out through the use of anOmmaya reservoir, in accordance with known techniques. (F. Balis et al.,Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989).

In some embodiments, the heparin-binding protein composition may beformulated to cross the blood brain barrier (BBB). One strategy for drugdelivery through the blood brain barrier (BBB) entails disruption of theBBB, either by osmotic means such as mannitol or leukotrienes, orbiochemically by the use of vasoactive substances such as bradykinin.Other strategies for transportation across the BBB may entail the use ofendogenous transport systems, including carrier-mediated transporterssuch as glucose and amino acid carriers, receptor-mediated transcytosisfor insulin or transferrin, and active efflux transporters such asp-glycoprotein. Alternatively, drug delivery behind the BBB is byintrathecal delivery of therapeutics directly to the cranium, as throughan Ommaya reservoir.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Heparin-Binding Protein Composition

The heparin binding protein composition of the present disclosureincludes heparin binding proteins secreted by pluripotent stem cells,such as, human embryonic stem cells. The compositions of the presentdisclosure may include heparin binding proteins isolated from a culturemedium conditioned by growth of pluripotent stem cells, such as, humanembryonic stem cells. In certain cases, the heparin binding proteincomposition may be a cell culture medium enriched for heparin bindingproteins, where the culture medium is conditioned by growth ofpluripotent stem cells.

Any appropriate method for isolating heparin binding proteins from aculture medium conditioned by growth of human embryonic stem cells maybe used. Exemplary methods include using heparin to bind to the heparinbinding proteins and isolating the bound heparin binding proteins. Theheparin may be conjugated to a solid support, such as, a column,membrane, nitro-cellulose, matrix, beads, and the like. The boundheparin binding proteins may be subsequently released from the heparinby disrupting the interaction between heparin binding proteins andheparin by using an elution solution that has a high or a low pH;choatropic agent, such as, a high salt concentration; detergents; etc.

A cell culture medium enriched for heparin binding proteins, where theculture medium is conditioned by growth of pluripotent stem cells may beprepared by removing non-heparin binding proteins from the cell culturemedium. For example, the conditioned medium may be processed to removeserum proteins such as albumin by using an antibody that binds toalbumin, thereby enriching the medium for heparin binding proteins.

In certain cases, the heparin binding protein composition may includeheparin binding proteins isolated from the conditioned growth medium andone or more proteins present in the conditioned growth medium. Forexample, the heparin binding protein composition may include heparinbinding proteins isolated from the conditioned growth medium and afraction of the conditioned growth medium. For example, the conditionedgrowth medium may be fractionated into different fractions where eachfraction contains proteins in a certain size range.

“Conditioned medium” refers to a growth medium that is furthersupplemented with soluble factors (“culture derived growth factors”)derived from pluripotent stem cells, such as iPS cells, ES cells, suchas, human embryonic stem cells or embryonic carcinoma cells, cultured inthe medium.

A conditioned medium may be a medium in which a pluripotent stem cell,such as iPS cells, ES cells, such as, human embryonic stem cells orembryonic carcinoma cells, has been grown for a period of 1 day-7 days.

In general, the heparin binding protein composition or a cell culturemedium enriched for heparin binding proteins secreted by pluripotentstem cells are essentially free of the pluripotent stem cells.Accordingly, the heparin binding protein composition or a cell culturemedium enriched for heparin binding proteins secreted by pluripotentstem cells do not have detectable numbers of pluripotent stem cells,such as, the pluripotent stem cell which secreted the heparin bindingproteins.

As noted herein, the heparin binding protein composition or a cellculture medium enriched for heparin binding proteins secreted bypluripotent stem cells may include one or more heparin binding proteinsthat enhance somatic cell proliferation and/or viability.

In general, the conditioned media is separated from the pluripotent stemcell prior to the isolation or enriching of heparin binding proteins.The separation by methods, such as, centrifugation, filtration,precipitation, results in a conditioned medium that is essentiallycell-free. In this context, “essentially cell-free” refers to aconditioned medium that contains fewer than about 10%, preferably fewerthan about 5%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001% than the number ofcells per unit volume, as compared to the culture from which it wasseparated.

In certain cases, the heparin binding protein composition may notinclude fibroblast growth factor(s) (FGF), such as, FGF-2. In certaincases, the cell culture medium enriched for heparin binding proteins maynot include fibroblast growth factor(s) (FGF), such as, FGF-2.

The medium conditioned may be conditioned from growth of any pluripotentstem cell, such as, induced pluripotent stem (iPS) cell, embryonic stem(ES) cell, embryonic carcinoma cells, human ES cell, human iPS cell,primate ES cell, primate iPS cell, mouse ES cell, mouse iPS cell, bovineES cell, bovine iPS cell, equine ES cell, equine iPS cell, and the like.

The heparin binding protein composition may be a formulation of theheparin binding protein(s) with an appropriate carrier or diluent. Inone aspect, heparin binding protein composition may be a formulation ofthe heparin binding protein(s) with an appropriate pharmaceuticallyacceptable excipient. The composition may be formulated into preparationin liquid or semi-solid or solid form, such as, solutions, injections,inhalants, gels, matrix, and the like.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available.

The subject methods are useful for both prophylactic and therapeuticpurposes.

The dosage of the formulation will vary widely, depending upon thenature of the condition, the frequency of administration, the manner ofadministration, the clearance of the heparin-binding protein from thehost, and the like. The initial dose can be larger, followed by smallermaintenance doses. The dose can be administered as infrequently asweekly or biweekly, or more often fractionated into smaller doses andadministered daily, semi-weekly, or otherwise as needed to maintain aneffective dosage level.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form”, refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of heparin-binding protein in an amount calculated sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particularformulation employed and the effect to be achieved, and thepharmacodynamics associated with formulation in the host.

In general, heparin-binding protein composition is administered at adose that is effective to cause an increase in somatic cellproliferation or enhance viability of a somatic cell, but whichmaintains the overall health of the individual. Treatment regimens willoften utilize a short-term administration of the heparin-binding proteincomposition; although the treatment may be repeated as necessary. Thetreatment regime can require administration for prolonged periods, butmay be administered as a single dose monthly, semi-monthly, etc. Thesize of the dose administered may be determined by a physician and willdepend on a number of factors, such as, the nature and gravity of theinjury, disease, the age and state of health of the patient.

Methods of the present disclosure may be practiced with a variety ofdifferent types of subjects. In the methods, the subject may vary. Incertain embodiments, the subjects are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g.,rabbits), and primates (e.g., humans, chimpanzees, and monkeys). Incertain embodiments, the subjects are humans.

Kits

The present disclosure also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of the heparinbinding protein compositions described herein. Kits may contain unitdoses of the heparin binding protein compositions. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Screening Methods

Methods for identifying modulators of somatic cell proliferation and/orviability are provided. Any somatic cell described herein may be used inthe screening methods.

In certain embodiments, a method for identifying modulators of somaticcell proliferation may include contacting a somatic cell with heparinbinding protein composition, wherein the composition comprises heparinbinding protein isolated from a medium conditioned by growth of humanembryonic stem cells, in the presence of a candidate agent wherein anincrease or decrease in the somatic cell proliferation compared to theabsence of the candidate agent identifies the candidate agent as amodulator of somatic cell proliferation.

In certain embodiments, the method may be for identifying candidateagents that increase somatic cell proliferation. In such methods, anincrease in the somatic cell proliferation compared to the absence ofthe candidate agent identifies the candidate agent as an enhancer ofsomatic cell proliferation.

Also provided are screening methods to identify candidate agents thatincrease viability of a somatic cell. The screening method may includecontacting the somatic cell with heparin binding protein composition,wherein the composition comprises heparin binding protein isolated froma medium conditioned by growth of human embryonic stem cells, in thepresence of the candidate agent, wherein increased viability of thesomatic cell as compared to the absence of the candidate agentidentifies the candidate agent as an enhancer of somatic cell viability.

Candidate agents of interest for screening include biologically activeagents of numerous chemical classes, primarily organic molecules,although including in some instances, inorganic molecules,organometallic molecules, immunoglobulins, genetic sequences, etc. Alsoof interest are small organic molecules, which comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, frequently at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules, including peptides,polynucleotides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Compounds may be obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds, including biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

A plurality of assays may be run in parallel with differentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in somatic cell proliferation/viability.

The screening methods may be carried out using any somatic cell ofinterest. The somatic cells may be as provided in the presentdisclosure.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Materials and Methods Animals

Young (2-3 month old) and old (22-24 month old) C57BL6/J mice werepurchased from the Jackson Laboratory and the NIH. The animalexperimental procedures were performed in accordance with the Guide forCare and Use of Laboratory Animals of the National Institutes of Health,and approved by the Office of Laboratory Animal Care, UC Berkeley.

Antibodies

Antibodies for phospho-ERK1/2, ERK1/2, and cleaved caspase 3 werepurchased from Cell Signaling. Laminin and Actin antibodies were fromSigma. FGF2 antibody was from Santa Cruz, Pax7 and eMyHC antibodies werefrom Hybridoma Bank, BrdU was from Abcam, and Map2 antibody was from BDBiosciences.

Muscle Fibers and Muscle Stem Cell Isolation

Uninjured TA muscle was dissected from healthy young and old mice andincubated at 37C in digestion medium (250 U/mL Collagenase type II inDMEM medium, buffered with 30 mM HEPES) for 1 hour (Bischoff, R.,Developmental biology, 1986. 115(1): p. 129-139). Digested muscle wasgently triturated and myofibers were collected. Myofibers were furtherdigested with 1 U/mL Dispase and 40 U/mL Collagenase type II to liberatemuscle stem cells (Conboy, M. J. and I. M. Conboy. Methods Mol Biol,2010. 621: p. 149-63). Muscle stem cells were cultured in DMEM withserum from the same age mouse.

Immunofluorescence Analysis

Cells were fixed with 4% PFA for 10 minutes before permeablization with0.1% Triton-X 100 for 30 minutes. Then cells were then immunostained forPax? (Hybridoma Bank) and ki67 (Abcam). Primary antibodies used forstaining cortical human neurons were: mouse anti-MAP2 (1:500, BDBiosciences), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling). Formuscle section immunostaining, an uninjured TA muscle was sectioned at10 um and stained for FGF2 (Santa Cruz) and laminin (Sigma).

Western blotting

Muscle stem cells or myofibers were lysed in RIPA buffer containing 1×protease inhibitor (Roche). The protein concentration was determined byBradford assay (Bio-Rad). Cell or fiber lysates were resuspended in 1×Laemmli buffer (Rio-rad), boiled for 5 minutes and separated on precastTGX gels from Biorad. The proteins were then transferred to PVDFmembrane (Millipore) and blotted with the desired antibodies.

Cell Culture

Rat NPCs were cultured in DMF12 (Gibco) with 5% N2 and 10 ng/mL FGF2, onlaminin and polyornithine coated plates. For experimental conditions,cells were plated at 40,000 cells/well in coated 8-well chamber slidesand cultured for 12-16 hours at 37 C in 10% CO2 incubator prior tofixation with 70% ethanol at 4 C. Adult human myoblasts were culturedand expanded in human growth medium (Ham's F-10 (Gibco), 10% BovineGrowth Serum (Hyclone), 30 ng/mL FGF2, and 1% penicillin-streptomycin onMatrigel (BD Biosciences) coated plates (1:100 matrigel:PBS), at 37 Cand 5% CO₂. For experimental conditions, cells were plated at 10,000cells/well in Matrigel coated 8-well chamber slides (1:100 Matrigel:PBS), and cultured for 72 hours with daily re-feedings at 37 C in 10%CO2 incubator prior to fixation with 70% ethanol at 4 C. Mouse myoblastswere cultured and expanded in mouse growth medium: Ham's F-10 (Gibco),20% Bovine Growth Serum (Hyclone), 5 ng/mL FGF2 and 1%penicillin-streptomycin on Matrigel coated plates (1:300 matrigel: PBS),at 37 C and 5% CO2. For experimental conditions, cells were plated at40,000 cells/well on Matrigel coated 8-well chamber slides (1:100matrigel: PBS) and cultured for 24 hours at 37 C in 10% CO₂ incubatorprior to fixation with 70% ethanol at 4 C.

Human embryonic stem cells (H9 and H7 lines), were cultured on dilutedMatrigel (1:30), in mTeSR-1 (Stem Cell Technologies), according tomanufacturer's recommendations. hESCs were differentiated after platingin mTeSR-1 by changing the medium to DMEM/F12 with 10% Bovine GrowthSerum (Hyclone), and culturing for an additional 6-8 days. Cells werewashed with Opti-MEM (Gibco) and then cultured in Opti-MEM for 18 hoursprior to collection as hESC-Conditioned Opti-MEM (hESC-ConditionedMedium) and stored at −80 C.

All experiments using a MEK inhibitor were treated with 10 micromolarMEK1/2 Inhibitor (U0126, Cell Signaling Technologies).

Cell Culture and Cortical Differentiation of Human Pluripotent StemCells

The H1 (WiCell) and hESC line was cultured on Matrigel-coated cellculture plates (BD) in mTeSR-1 maintenance medium (Stem CellTechnologies). In adherent conditions, hPSCs were seeded at a density of5×104 cells/cm2 in growth medium. At 50% confluence, the medium wasgradually changed to neural basal medium (Invitrogen) containing N2 andB27 (Invitrogen). SMAD signaling inhibitors LDN193189 (Stemgent, 1 μM)and SB432542 (Tocris Biosciences, 10 μM) were added from day 1 to day 7of neural induction. Cyclopamine (Calbiochem, 400 ng/ml) and FGF-2(Peprotech, 10 ng/ml) were added from days 3-14 of differentiation.After 12-14 days, cells were mechanically passaged into poly-L-ornithine(Sigma Aldrich) and laminin (Invitrogen, 20 μg/ml) coated plates andallowed to undergo maturation for 3-6 weeks. BDNF (10 ng/ml, Peprotech)was added to cultures one week after initiation of neuronal maturation.For EB mediated neural differentiation, PSCs were aggregated for 4 daysin ultra low-attachment plates (Corning) and then seeded onMatrigel-coated plates. Cyclopamine (5 μM) and FGF-2 (10 ng/ml) wereadded to the cultures the following day until day 12 of neuralinduction. At day 14, structures with a rosette-like morphology weremechanically isolated and plated on poly-L-ornithine and laminin coatedplates and allowed to undergo neuronal maturation for 4 weeks. BDNF (10ng/ml) was added to the cultures one week after rosette isolation.

Globulomer Preparation

The Aβ42 globulomer was prepared as described (Barghorn et al., 2005, JNeurochem. 95, 834-47; Yu et al., 2009, Biochemistry. 48, 1870-7).Alkaline pretreatment of Aβ42 and preparation of low molecular weight Aβby filtration protocols were used before beginning the globulomerpreparation as previously described (Yu et al., 2009, supra). After the18-20 h incubation, the globulomer sample were concentrated to ˜500 mMvia centrifugation and dialyzed into PBS before centrifuging the sampleat 10,000 g for 10 min to remove aggregates in the pellet. Thesupernatant was saved, and the absorbance was measured at 276 nmwavelength to measure the concentration (extinction coefficient=1390 M−1cm−1).

Immunocytochemistry

For immunoflourescence assays, human and mouse myoblasts were given a 4hour and 2 hour 300 μm BrdU pulse, respectively. Cells were thenpermeabilized in PBS +0.25% Triton X-100 and incubated with primaryantibodies overnight at 4° C. in PBS +2% FBS. Antigen retrieval wasperformed via a 10 minute 4 N HCl treatment followed by PBS wash.Primary staining was performed overnight with species-specificmonoclonal antibodies for mouse anti-embryonic Myosin Heavy Chain(eMyHC) (eMHC hybridoma (clone 1.652), Developmental Studies HybridomaBank) and Rat-BrdU (Abcam Inc. ab6326) for myoblasts, and Goat-Sox2(Santa Cruz) for rNPCs. Secondary staining with fluorophore-conjugated,species-specific antibodies (Donkey anti-Rat-488, #712-485-150; Donkeyanti-Mouse-488, #715-485-150; Donkey anti-Rat-Cye3 #712-165-150; ordonkey anti-Mouse-Cye3 #715-165-150; all secondary antibodies fromJackson ImmunoResearch) was performed for 1 hour at room temperature ata 1:500 dilution in PBS +2% FBS. Nuclei were visualized by Hoechststaining, and samples were analyzed at room temperature with a ZeissAxio Imager Al, and imaged with an Axiocam MRC camera and AxioVisionsoftware. Human and mouse myoblasts were imaged at 10× and 20×magnification, respectively.

For cell quantification, 25-50 20× images per replicate were taken onthe Molecular Devices ImageXpress Micro automated epifluorescenceimager, followed by automated cell quantification using themultiwavelength cell scoring module within the MetaXpress analysissoftware.

Heparin Bead Depletion of hESC-Secreted Proteins from hESC-ConditionedMedium

Heparin-Agarose Type I Beads (H 6508, Sigma Aldrich) were washed withmolecular grade water and preconditioned in 1 mL OptiMEM as recommendedby manufacturer. hESC-conditioned medium was incubated withHeparin-Agarose Beads for 2 hours shaking at 4° C. Beads and all mediumwere separated by centrifugation. Myoblasts were treated with depletedmedium after two rounds of centrifugation and separation of beads andmedium so as to remove all residual beads from depleted hESC-conditionedmedium.

Heparin Bead Bound Proteins Elution and Purification

After depleting hESC-Conditioned OptiMEM, the protein bound heparinbeads were washed two times for 10 minutes at 4° C. in 1 ml PBS +0.05%Tween. Proteins were eluted twice for 15 minutes at 4° C. in 400 μl ofelution buffer (0.01M Tris-HCl pH 7.5+1.5M NaCl+0.1% BSA) to collectproteins in a total of 800 μl of elution buffer. The proteins werepurified by diffusion dialysis by a 2 hour dialysis shaking at 4° C. in500 ml McCoy's 5A Medium (Gibco) followed by overnight dialysis shakingat 4° C. in 200 ml OptiMEM (Gibco). The eluted heparin beads werere-suspended in 800 μl OptiMEM and stored overnight at 4° C. One hourafter plating, mouse myoblasts were treated with respective mediums for24 hours prior to 2 hour BrdU pulse and fixation in 70% ethanol.

Muscle Injury

Isoflurane was used to anesthetize the animal during the muscle injuryprocedure.

For bulk myofiber satellite cell activation, gastrocnemius muscles wereinjected with cardiotoxin 1 (Sigma) dissolved at 100 micrograms permilliliter in PBS, at 4 sites of 10 microliters each for each muscle.Muscles were harvested 3 days later. For focal injury, to assayregeneration in vivo, 5 microliters of 0.5 milligram per milliliter CTXwas injected at two sites to the middle of the tibialis anterior, andmuscle harvested 5 days later.

Tissue Immunofluorescence and Histological Analysis

Muscle tissue was dissected, flash frozen in OCT compound (Tissue Tek;Sakura) and cryo-sectioned at 10 micrometers, as previously described(Conboy et al., 2003). Cryo-sectioning was performed through the entirevolume of muscle (typically 50-70 sections total, done at 200 μmintervals), thereby serially reconstituting the entire tissue, ex vivo.Muscle sections were stained with aqueous hematoxylin and eosin (H&E),as per the manufacturer's instructions (Sigma-Aldrich). Regeneration andmyogenic potential was quantified by examining injury sites fromrepresentative sections along the muscle (spanning the volume ofinjury), then by measuring the injured/regenerating area using AdobePhotoshop Elements. Myofiber regeneration was quantified by countingtotal newly regenerated fibers and dividing by the regeneration area.Immunostaining was performed as described (Conboy, M. J., et al.,Methods Mol Biol, 2010. 621: p. 165-73). Briefly, after permeabilizationin PBS +1% FBS +0.25% Triton-X-100, tissues and cells were incubatedwith primary antibodies in staining buffer (PBS +1% FBS) for 1 h at roomtemperature, followed by 1 h incubation fluorochrome-labeled secondaryantibodies (ALEXA at 1:1000). BrdU-specific immunostaining required anextra step of 2 M HCl treatment before permeablization.

Example 1 mTeSR-1 Growth Medium has Pro-Myogenic Activity, Which is Dueto the High Levels of FGF-2, and hESC-Secreted Factors Act Independentlyof Recombinant FGF-2

Our previous work established that injection of hESCs—which werecultured on mouse embryonic fibroblasts (MEF) and in standard, highlymitogenic, embryonic cell growth medium—enhanced old muscle regeneration(Carlson, M. E. and I. M. Conboy, Aging Cell, 2007. 6(3): p. 371-82). Inour more recent work, the hESCs have been cultured in mTeSR-1 (Stem CellTechnologies), a defined feeder-free medium which is also highlymitogenic (Ludwig, T. E., et al., Nat Methods, 2006. 3(8): p. 637-646),and we investigated whether and to what degree the pro-myogenic effectsof hESC-conditioned medium was due to the residual activity of the hESCgrowth/expansion medium. Primary muscle progenitor cells (myoblasts)were cultured overnight in a mitogen-low fusion medium that typicallyinduces differentiation of myoblasts into multinucleated eMyHC+myotubes. The enhancement of myogenic cell proliferation and inhibitionof differentiation was assayed by BrdU uptake for the last 2 hours ofculture, after which cells were fixed and used for immunofluorescencewith anti-BrdU and anti-MyHC specific antibodies. When primary myoblastswere cultured in 50% fusion medium plus 50% hESC-conditioned mTeSR-1 or50% unconditioned mTeSR-1, both media compositions induced proliferationand inhibited differentiation of these myogenic cells, though mediumcontaining hESC-conditioned mTeSR-1 inhibited differentiation moresignificantly (FIG. 1A, quantified in 1B and 1C). To confirm these datawith muscle stem cells, injury-activated satellite cells associated withmyofibers were isolated from old muscle and cultured overnight in a50/50 mix of Opti-MEM containing 5% old mouse serum and hESC-conditionedmTeSR-1 or mTeSR-1. Both conditioned and not-conditioned mTeSR-1 mediaenhanced the regenerative capacity of satellite cells that were isolatedfrom injured old muscle, based on the numbers of de-novo generatedBrdU+/Desmin+ muscle progenitor cells (FIG. 1D, quantified in 1E). Theseresults demonstrate that embryonic stem cell culture medium itself haspro-myogenic effects. To investigate whether hESC-conditioned Opti-MEMexhibits pro-regenerative effects due to the hESC-secreted proteins, andnot because of residual mTeSR-1, we washed the hESC culture wellsmultiple times with Opti-MEM prior to incubation for conditioning theOpti-MEM, and found that even after 3 washes, hESC conditioned theOpti-MEM to yield the same potent pro-regenerative effect on myoblasts(FIG. 1F). These results demonstrate that while mTeSR-1 supplementationpromotes myoblast proliferation, other factor(s) produced by hESCsindependently enhance the regenerative capacity of muscle stem andprogenitor cells.

To understand the pro-myogenic effects of mTeSR-1 in greater detail, weaddressed the role of FGF-2, which is present at high concentration inmTeSR-1 (over 50 nanograms per milliliter, ˜10 times higher than thedoses used in conventional culture of muscle progenitor cells). Ourhypothesis was that the FGF-2 in mTeSR-1 enhances myoblast and satellitecell proliferation, partially masking the effects of the hESC-producedfactors in hESC-conditioned mTeSR-1. To test this hypothesis, weincubated hESCs in a basal medium that had the other growth andsignaling factors present in mTeSR-1 (TGF-beta, GABA, pipecolic acid andLithium Chloride, (Ludwig, et al, supra)), but lacked FGF2, and comparedthe pro-myogenic effects of this FGF-free hESC-conditioned mTeSR-1analog with the effects of the same mTeSR-1 analog that was notconditioned by the hESCs. Without FGF-2, the mTeSR-1 analog lackedpro-regenerative effects on myoblasts (FIG. 2A, quantified in 2B). Onthe other hand the very same mTeSR-1 analog lacking FGF-2, butconditioned by hESCs, significantly enhanced myoblast proliferation andinhibited differentiation, while conditioning of this mTeSR-1 analoglacking FGF-2 by differentiated hESC derived cells resulted in theabsence of pro-myogenic properties (FIG. 2A, quantified in 2B). Thesedata demonstrate that the pro-myogenic effects of mTeSR-1 are due to thehigh concentration of FGF-2, and that it is not simply residual FGF-2from mTeSR-1 that is responsible for the enhancement of myogenesis bythe hESC-conditioned medium.

Example 2 FGF-2 Signaling and Satellite Cell Proliferation are notIncreased with Age

FGF-2, which often functions as a mitogen, was recently reported tocontribute to the aging and depletion of mouse satellite cells. However,the canonical model of muscle stem cell aging postulates that a declinein such mitogens over time leads to reduced activation of satellitecells that are resident to old tissue (Conboy, I. M. and T. A. Rando,Cell Cycle, 2012. 11(12): p. 2260-7; Grounds, M.D., Ann. N.Y. Acad.Sci., 1998. 854: p. 78-91; onboy, I. M. and T. A. Rando, Cell Cycle,2005. 4(3): p. 407-410), so we explored these phenomena in more detail.The levels of FGF-2 were determined by Western Blotting in muscle fibersthat were derived from Tibialis Anterior (TA) and Gastrocnemius(Gastroc) muscle of young and old mice. As shown in FIG. 3A (quantifiedin 3B), a significant increase in FGF-2 protein was observed with age inmyofibers, consistent with Chakkalakal et al. FGF-2 signals through theMAPK/pERK pathway, so we analyzed the levels of pERK in myofibersderived from young and old uninjured muscle. Interestingly, as shown inFIG. 3A (quantified in 3B), no age-specific increase in pERK was found,and the levels of this key effector were very low in cells from bothages, despite the high levels of FGF-2 in protein lysates derived fromold muscle fibers. Also, a myoblast control indicates that pERKdetection was sensitive (FIG. 3A). To understand these data, we examinedthe presence and localization of FGF-2 in the intact young and oldmuscle, using 10 micron cryosections. FGF-2 and laminin were detectedwith specific antibodies and resolved by immunofluorescence. As shown inFIG. 3 C (quantified in 3D), FGF-2 was localized in the basementmembrane of young muscle, while in the old muscle, FGF-2 was presentless in the basement membrane and more in the cytoplasm of the myofibers(e.g. away from its receptors in muscle stem cells). These data suggestthat the relatively higher levels of FGF-2 in old muscle do notnecessarily represent ligand that is available for signaling insatellite cells. Additionally, these results indicate that detection ofelevated FGF-2 in the old muscle might be due to its over-expressionwithin the old muscle fiber itself, or alternatively, due to “washing”of extracellular FGF-2 from young muscle during tissue dissociation whenthe basement membrane is digested with collagenase and dispase, andtissue integrity is perturbed (Bischoff, R., Developmental biology,1986. 115(1): p. 129-139).

To confirm and extend upon these findings, we isolated muscle stem cellsfrom uninjured young and old TA and Gastroc muscle and treated them withFGF-2 for 30 minutes, after which the levels of FGF-2, pERK, and totalERK were determined in these freshly isolated stem cells. As shown inFIG. 4 A, B, endogenous FGF-2 was undetectable in either young or oldmuscle stem cells upon isolation, but the added FGF-2 was clearlypresent in these satellite cells after 30 minutes. Young and oldsatellite cells were harvested after just 30 minutes of culture, thus,the FGF-2 protein detected in cultures, which were treated withrecombinant FGF-2 is unlikely to represent de-novo expression. Satellitecells were lifted from the plates with PBS and washed prior to theirlysing for Western Blotting, and it was thus unlikely that any residual,non-cell associated recombinant FGF-2 from media or plates wouldcontaminate cell lysates. To test this directly and definitively, weperformed a control with a matrix-coated but cell-free plate that wasidentically treated with FGF-2, and found no detectable recombinantFGF-2 in the solution (FIG. 4A). Hence, the FGF-2 detected in proteinlysates of young and old satellite cells incubated with this growthfactor likely reflects ligand that is bound to its specific receptors.In support of this conclusion, recombinant FGF-2 induced pERK in bothyoung and old satellite cells (FIG. 4 A and C). In agreement withnon-detectable endogenous FGF-2 in both young and old satellite cells,very low levels of pERK that did not differ with age were observed inthese muscle stem cells resident to tissue that was neither injured nortreated with recombinant FGF-2 (FIG. 4 A and C). To determine whetherlow levels (as opposed to none) of FGF-2 can be detected in the musclestem cells, another independent experiment was performed with aprolonged enhanced chemiluminescence exposure of the Western Blots. Asshown in FIG. 5, low levels of FGF-2 could be indeed detected in musclestem cells after a 30 minute exposure, but once again, there was noage-specific difference in either FGF-2 or in pERK. These resultssuggest that FGF-2 does not signal in either young or old satellitecells that reside in non-injured skeletal muscle.

To directly examine cell proliferation, satellite cells were isolatedfrom non-injured young and old tissue and were cultured with or withoutFGF-2 overnight, after which the levels of the proliferation marker Ki67were determined in Pax7+ satellite cells. Muscle stem cells for this andother experiments were isolated with high and equal purity from youngand old mice, as shown in FIG. 6. Neither young nor old cells were lostduring overnight culturing, as the numbers were similar to initialplating, and no age-specific loss was observed, based on the cellcounts. As shown in FIG. 4 D and E, no increase in proliferation of agedmuscle stem cells was detected, as compared to young, and as expectedfrom previous literature, the majority of both young and old satellitecells were quiescent Conboy, I. M., et al.,. Science, 2003. 302(5650):p. 1575-1577; Bischoff, R., Developmental biology, 1986. 115(1): p.129-139; Conboy, I. M. and T. A. Rando, Dev. Cell, 2002. 3(3): p.397-409. When added, FGF-2 significantly enhanced the proliferation ofquiescent muscle stem cells that were isolated from uninjured muscle(both young and old), as shown in FIG. 4 D and E, which is consistentwith the induction of pERK that is shown in FIG. 4 A, C. However, veryinterestingly, 90-95% of muscle stem cells derived from uninjured youngand old tissue were not proliferating even in the presence of addedFGF-2, suggesting that other mitogens and/or cell-fate changes areneeded to induce the robust entry of quiescent satellite cells into thecell cycle, also as published (Bischoff, R., J Cell Biol, 1990. 111(1):p. 201-7). These data demonstrate that the localization of FGF-2 withinthe skeletal muscle compartment changes with age and question whetherendogenous FGF-2 is likely to exhaust the pool of aged quiescentsatellite cells, since it does not induce significant signaling in thesecells.

Example 3 The Pro-Regenerative Activity of hESC-Secreted Factors isContained in Proteins with Heparin Binding Domains

To confirm that the factors in hESC conditioned medium were proteins,hESC conditioned Opti-MEM was treated with proteinase-K agarose beads,and the beads were removed before mixing 50/50 with Opti-MEM and 5%mouse serum, for culture with injury-activated satellite cells withassociated fibers from old muscle, as above. All proliferative activityof the conditioned medium was lost after proteinase treatment,indicating that protein(s) conferred the pro-regenerative activity (FIG.7).

To deplete heparin-binding proteins, hESC-conditioned medium wasincubated with heparin binding domain-coated acrylic beads. Muscleprogenitor cells were then cultured in this heparin-depletedhESC-conditioned medium, hESC-conditioned medium, or controls (mediumalone and medium conditioned by differentiated cells that lack thepro-regenerative activity). Proliferation of primary muscle progenitorcells was assayed by BrdU uptake for 2 hours, and cell differentiationwas assayed by the expression of eMyHC. Interestingly, hESC-conditionedmedium depleted of heparin binding proteins completely lost itspro-regenerative activity on muscle progenitor cells (FIG. 8 A,quantified in B). Even more importantly, the pro-regenerative activityof in the hESC-secreted proteins could be eluted from the heparin-coatedbeads (FIG. 8 A, quantified in 8B), hence confirming that these factorshave heparin-binding domains and suggesting novel strategies forpurification of these clinically relevant molecules. Excitingly, whenthese heparin-binding eluted embryonic proteins were injected at Day 0and Day 2 into injured muscle (e.g., at the time of the injury and whenmuscle stem cells become activated for regeneration) old muscle repairbecame rejuvenated, based on increased formation of de-novo myofiberswith centrally located BrdU+ nuclei (FIG. 8C, quantified in 8D). Thesedata reveal the pro-myogenic proteins that are secreted by the hESCscontain heparin-binding domains.

Example 4 hESC-Conditioned Opti-MEM has Pro-Survival and Pro-MitogenicEffects on Neuronal Cell Types

To assess the potential positive effect of hESC-secreted proteins onother cell types, specifically neural cells, we cultured rat neuralprogenitor cells in the presence of hESC-conditioned medium, or in acontrol medium conditioned by differentiated hESC-derived cells.Specifically, cells were cultured in the 50/50 mix of neuraldifferentiation medium (see Methods) and Opti-MEM, which was conditionedeither by the self-renewing hESCs or by the negative control,differentiated hESC-derived cells. The goal was to determine ifhESC-secreted factors can enhance proliferation and inhibitdifferentiation of NPCs, in parallel to our studies demonstrating theseembryonic factors enhance muscle precursor proliferation and inhibittheir differentiation in a 50/50 mix of fusion medium (Conboy, I. M., H.Yousef, and M. J. Conboy, Aging (Albany NY), 2011. 3(5): p. 555-63).Very interestingly, a significant increase in proliferation of Sox-2+neural progenitors was observed in cultures exposed to the hESC-producedproteins, an effect that was lost when NPCs were cultured in controlmedium from differentiated cells (FIG. 6 A, quantified in B). As thiseffect was similar to what we previously reported for musclestem/progenitor cells, in that we observe an enhancement ofproliferation and inhibition of differentiation of precursor cells byhESC-secreted factors (Conboy, I. M., H. Yousef, and M. J. Conboy, Aging(Albany N.Y.), 2011. 3(5): p. 555-63), it suggests that hESC-secretedproteins enhance the proliferative capacity of progenitor cells inmultiple tissue types, and similarly to the situation in muscle, thepro-mitogenic activity is lost when hESCs differentiate.

We next sought to examine whether not only cell proliferation, but cellviability might be enhanced by the hESC-secreted proteins, particularlyunder pathological conditions. Likewise, we wished to investigatewhether the effects of the pro-mitogenic factors would manifest not onlyon progenitors, but also on terminally differentiated neurons. To answerthese questions, we generated human cortical glutamatergic neurons bydirected differentiation of embryonic stem cells (see Methods).Specifically, dorsal telencephalic progenitors expressing glutamate andVgluT1 were generated by using Shh and FGF-2. This protocol induced thedifferentiation of human embryonic stem cells (hESCs) into cultures withup to 74% of neurons expressing glutamate and VgluT1. As an in vitromodel of AD, soluble oligomeric forms of Aβ known as “globulomers,”which have been implicated in the pathology of Alzheimer's disease (Kuo,Y. M., et al., J Biol Chem, 1996. 271(8): p. 4077-81; Jensen, M., etal., Mol Med, 2000. 6(4): p. 291-302), were added to these cultures ofhuman glutamatergic neurons. They bound Aβ, which led to cell death asmeasured by the presence of cleaved caspase 3 (FIG. 9C, quantified in9D).

To examine whether hESC-secreted factors have neuroprotective effects inthis in vitro human AD model, Aβ globulomers were added to corticalcultures primarily comprised of glutamatergic neurons in the presence orabsence of hESC-conditioned medium. The neurons were pre-incubated withhESC-conditioned Opti-MEM for 1 hr prior to treatment with Aβglobulomers, or alternatively, hESC-conditioned medium was added at 50%to neuron medium, simultaneously with the Aβ globulomers. Analysis withcleaved caspase-3 as an apoptotic marker and MAP2 as neuron markershowed a significant decrease in cell death when neurons werepre-incubated with hESC-secreted factors, as compared to culturestreated with Aβ globulomers alone (FIG. 9C, quantified in 9D). Anoticeable but not statistically significant decrease of apoptosis wasobserved in neuron cultures that were administered with Aβ andhESC-secreted proteins simultaneously (FIG. 9 C, D). These data suggestthat hESC-secreted factors exert a protective (anti-apoptotic) effect onhuman cortical neurons in this AD model.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for enhancing proliferation of a somaticcell, the method comprising: contacting a somatic cell with heparinbinding protein composition, wherein the composition comprises heparinbinding protein isolated from a medium conditioned by growth of humanembryonic stem or carcinoma cells, wherein the contacting is for aperiod of time sufficient to provide for enhanced proliferation of thesomatic cell compared to the absence of the heparin binding proteincomposition.
 2. The method of claim 1, wherein the somatic cell is cellof an aged subject.
 3. The method of claim 1, wherein the somatic cellis an injured cell.
 4. The method of claim 3, wherein the injury iscaused by a disease.
 5. The method of claim 1, wherein the somatic cellis a diseased cell.
 6. The method of claim 1, wherein the somatic cellis a muscle cell.
 7. The method of claim 6, wherein the muscle cell is askeletal muscle cell.
 8. The method of claim 7, wherein the skeletalmuscle cell is a myoblast.
 9. The method of claim 1, wherein the cell isan activated satellite cell.
 10. The method of claim 1, wherein thesomatic cell is a neural cell.
 11. The method of claim 10, wherein theneural cell is a neural stem cell.
 12. The method of claim 10, whereinthe neural cell is a neural progenitor cells.
 13. The method of claim 1,wherein the somatic cell is an adult human stem or an adult humanprogenitor cell.
 14. The method of claim 1, wherein the heparin bindingprotein composition comprises a plurality of heparin binding proteinsisolated from a medium conditioned by growth of human embryonic stemcells.
 15. Use of a heparin binding protein composition, wherein thecomposition comprises heparin binding protein isolated from a mediumconditioned by growth of human embryonic stem cells, for enhancingproliferation of a somatic cell by contacting the somatic cell with theheparin binding protein composition for a period of time sufficient toprovide for enhanced proliferation of the somatic cell as compared tothe absence of the heparin binding protein composition.
 16. A method ofincreasing viability of a neuron, the method comprising: contacting theneuron with heparin binding protein composition, wherein the compositioncomprises heparin binding protein isolated from a medium conditioned bygrowth of human embryonic stem cells, wherein the contacting is for aperiod of time sufficient to provide for increasing viability of theneuron as compared to the absence of the heparin binding proteincomposition.
 17. The method of claim 16, wherein the neuron is acortical neuron.
 18. The method of claim 16, wherein the neuron is aglutamatergic neuron.
 19. The method of claim 16, wherein the neuron isexposed to a toxin.
 20. The method of claim 16, wherein the toxin isamyloid beta globulomer.
 21. Use of a heparin binding proteincomposition, wherein the composition comprises heparin binding proteinisolated from a medium conditioned by growth of human embryonic stemcells, for increasing viability of a neuron by contacting the neuronwith the heparin binding protein composition for a period of timesufficient to provide for increasing viability of the neuron as comparedto the absence of the heparin binding protein composition.